THE CYTOCHROME BC(1) COMPLEX BUT NOT CYCM IS NECESSARY FOR SYMBIOTIC NITROGEN-FIXATION BY RHIZOBIUM-LEGUMINOSARUM

Following Tn5 mutagenesis of Rhizobium leguminosarum biovar viciae, tw o mutants in one complementation group were identified as being unable to fix nitrogen in pea nodules, Spectroscopic analysis revealed that the mutants had lowered levels of c-type cytochromes and cytochromes a a(3), but increased levels of cytochrome d. Cells of the mutants were greatly reduced in their ability to oxidize the artificial electron do nor N, N, N', N'-tetramethyl-p-phenylenediamine but membranes prepared from them had increased levels of succinate- and NADH-dependent respi ration. NADH oxidation by the mutants was insensitive to the respirato ry inhibitor antimycin A, that targets the cytochrome bc(1) complex. M olecular analysis of the mutants revealed that they were affected in t he cytochrome bc(1) complex. One of the mutants contained Tn5 in a gen e homologous to that encoding cytochrome c(1), and in the other the Tn 5 was in DNA homologous to that encoding the cytochrome b component of the cytochrome bc(1) complex, Haem staining revealed that haem protei ns of M(r) 31000 and M(r) 23000 were absent from membranes from the mu tants whereas an additional soluble c-type cytochrome protein of M(r) 23000 was present. We conclude that the larger of these two haem prote ins corresponds to cytochrome c, and, in its absence, the protein of M (r) 23000 does not remain associated with the membrane. Formation of t his M(r) 23000 component was specifically blocked in a third respirato ry-defective mutant which contained Tn5 in a region of DNA showing hom ology to a Bradyrhizobium japonicum gene previously shown to encode th e membrane-bound c-type cytochrome CycM, Although the cytochrome be, c omplex is essential for symbiotic nitrogen fixation, the other membran e-bound c-type cytochrome (CycM) is not.